Genetic manipulations at the DNA level and RNA interference (RNAi) have been the mainstream tools for functional studies of proteins. However, inducible protein knockout has a number of advantages over these traditional approaches, including the potential to induce fast and reversible response by the ligand, tunability (dosage-dependent quantitative depletion), depletion of the target protein from a specific cellular pool only, and depletion of a specifically-modified form of the target protein. However, a general inducible protein knockout system that can deplete specific endogenous protein(s) remains to be developed. Most recently, we have defined the first molecular mechanism for allosteric activation of E3 ubiquitin ligases. RNF146 is an intrinsically inactive E3 in the absent of a ligan, and becomes robustly activated in the presence of its ligand (iso-ADPr, a structural unit of poly (ADP-ribose)). With well-defined molecular mechanism and structural basis for this allosteric switch, we aim to use RNF146/iso-ADPr as the template to develop an inducible, tunable protein knockout system. The bottle neck for this development is to design cell-permeable iso-ADPr orthologue(s) and engineer the RNF146 protein accordingly, so that the engineered protein responds to the orthologue compound but not to the endogenous iso-ADPr moiety in PAR polymers. In this project, we will overcome this bottleneck and validate the desired binding and activation specificities for our co-engineered orthogonal protein/ligand pairs, using the depletion of endogenous ?-catenin, a central effector of Wnt signaling, as a model system. Development and optimization of a general inducible protein knockout system will not only provide a powerful tool for biological research, but may also have profound implications in medicine.